Direct amplification and detection of viral and bacterial pathogens

ABSTRACT

Provided herein are methods for identifying the presence or absence of a target nucleic acid from a microorganism using direct amplification without a step of extraction of the nucleic acids, but retaining substantially the same specificity and sensitivity of methods assaying extracted nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/130,705, filed May, 2014, now U.S. Pat. No. 9,464,331, which is aU.S. national stage of PCT/US2012/045763 filed Jul. 6, 2012, whichclaims the benefit under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication No. 61/505,055, filed Jul. 6, 2011 and U.S. ProvisionalApplication Ser. No. 61/552,405, filed Oct. 27, 2011. The disclosures ofeach of the prior applications are considered part of and areincorporated by reference in the disclosure of this application.

FIELD OF THE INVENTION

The present invention relates to diagnostic and detection methods forviral and bacterial pathogen nucleic acids using direct amplification.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Clinical detection of viruses is usually accomplished using any one of avariety of methods. For example, virus particles or nucleic acids may beisolated from a biological sample (e.g., nasopharyngeal aspirates,throat swabs, blood fluids, fecal material, etc.). A retrospectivediagnosis may be made by serology. Complement Fixation Tests (CFT) aremost widely used in this method, although hemagglutination inhibition(HAI) and enzyme immunoassays (EIA) may be used to give a type-specificdiagnosis. For more rapid diagnosis, either antigen detection or RNAdetection may be performed. Antigen detection may be done by IFT or EIA,however, to achieve the highest level of sensitivity and specificity,RNA detection by reverse transcriptase polymerase chain reaction(RT-PCR) is used. However, the latter is expensive and technicallydemanding.

Similarly, bacterial detection may be accomplished using a variety ofmethods, including gram staining, culture, microarray, and polymerasechain reaction (PCR) or real-time PCR. Unlike detection of viruses suchas Influenza, which has a genome composed of RNA and therefore requiresa transcription step to create target cDNA for use in traditional orreal-time PCR, bacterial detection can be accomplished using a standardPCR protocol. Even without the additional RT step, however, PCR orreal-time PCR are, as described below, time-consuming and expensivediagnostic methods.

RT-PCR is a laboratory technique used to amplify and quantify a targetednucleic acid. The procedure follows the general principle of polymerasechain reaction, although in RT-PCR an RNA strand is first reversetranscribed into its DNA complement (cDNA) using the enzyme reversetranscriptase, and the resulting cDNA is amplified using traditional PCRor real-time PCR. The reverse transcription (RT) step can be performedeither in the same tube with PCR (one-step PCR) or in a separate one(two-step PCR) using a temperature between about 40° C. and 50° C.,depending on the properties of the reverse transcriptase used. The dsDNAis then denaturized at about 95° C., so that the two strands separateand the primers can bind again at lower temperatures and begin a newamplification reaction. DNA extension from the primers takes place usinga thermostable Taq DNA polymerase, usually at about 72° C. Real-timeRT-PCR provides a method in which the amplicons can be visualized as theamplification progresses using a fluorescent reporter molecule.

Given the high degree of complexity associated with the preparing andprocessing viral and bacterial nucleic acids from biological samples fordetection, diagnosis, and/or quantitation, in cases where rapiddiagnosis is sought, there is a need for methods involving fewer steps,fewer technological requirements, and shorter durations.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of a reagent mixturethat allows for direct amplification of a sample, without the step ofnucleic acid extraction.

In one aspect, the present invention provides a method for identifyingthe presence or absence of a target nucleic acid from a microorganism ina biological sample obtained from a human, said method comprising: (a)contacting the sample with a DNA polymerase and a buffer underconditions suitable for amplification of the target nucleic acid fromthe sample without extracting the target nucleic acid from the sample;(b) thermocycling the sample from step (a) such that the target nucleicacid, if present, is amplified; and (c) detecting the amplified targetnucleic acid, if present, produced from step (b), wherein the samplenucleic acid is not extracted prior to amplification.

In another aspect, the present invention provides a method foridentifying the presence or absence of a target nucleic acid from amicroorganism in a biological sample obtained from a human, said methodcomprising: (a) contacting the sample with a DNA polymerase and a bufferunder conditions suitable for amplification of the target nucleic acidfrom the sample without extracting the target nucleic acid from thesample; (b) thermocycling the sample from step (a) such that the targetnucleic acid, if present, is amplified; and (c) detecting the amplifiedtarget nucleic acid, if present, produced from step (b), wherein nucleicacid in the sample is not extracted from the sample prior toamplification, and wherein the buffer comprises at least one ofcomponent selected from the group consisting of KCl, bovine serumalbumin and a surfactant.

In some embodiments, the sample nucleic acid is not diluted prior tostep (a). In further embodiments, the sample may be heated prior to step(b), or, in still further embodiments, prior to step (a). The sample maybe heated prior to step (b) for at least about 2 minutes at atemperature of at least about 70° C.

In further embodiments, the buffer may comprise potassium chloride(KCl), and, in some embodiments, KCl may be present in a concentrationof about 5 mM to about 50 mM. The buffer may further comprise the GOTAQ®Flexi Buffer (Promega, Madison, Wis.), which may be present during step(b) in a 1×-5× concentration. The buffer may also comprise bovine serumalbumin. The buffer may comprise a surfactant. In some embodiments, thesurfactant is a cationic surfactant. In still further embodiments, theDNA polymerase is a Taq polymerase. The target nucleic acid may be DNAor, in some embodiments, RNA. When the sample is an RNA, it may befurther contacted with a reverse transcriptase. The sample may besimultaneously contacted with the DNA polymerase and the reversetranscriptase.

In further embodiments, the sample is selected from the group consistingof blood, serum, plasma, cerebrospinal fluid, oral fluid, and stool. Insome embodiments, the sample is whole blood. In some embodiments, thesample is obtained from the buccal region. In still further embodiments,the microorganism may be a virus, or may be selected from the groupconsisting of an influenza virus, a respiratory syncytial virus, aherpes simplex virus, and an enterovirus. The microorganism may also, insome embodiments, be a bacterium, such as, in further embodiments, C.difficile.

As used herein, the term “RNA” refers to a nucleic acid moleculecomprising a ribose sugar as opposed to a deoxyribose sugar as found inDNA. As used herein, RNA refers to all species or RNA includingmessenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), as wellas small RNA species that have regulatory function. “Small RNA species”have a specific meaning and refer to untranslated RNAs with housekeepingor regulatory roles in bacteria. “Small RNA species” are not rRNA ortRNA.

As used herein, the term “target nucleic acid” refers to any nucleicacid molecule or fragment that is diagnostic of a particular virus orbacteria including, for example, a pathogen virus or bacterial. Targetnucleic acids may be DNA or RNA molecules that are derived from thetarget species.

As used herein, the term “thermocycling” refers to any technique bywhich a laboratory apparatus is used to amplify segments of nucleic acidwith a primer extension reaction using pre-programmed cycles of raisedand lowered temperatures. Examples of thermocycling include, but are notlimited to, PCR, real-time PCR, and RT-PCR.

As used herein, the term “reverse transcriptase polymerase chainreaction” or “RT-PCR” refers to any technique for synthesizing andamplifying a DNA molecule with a sequence that is a copy of an RNAsequence. RT-PCR is useful in detecting RNA species such as inquantitative analysis of gene expression, as well as for producing DNAcopies of RNA for use in cloning, cDNA library construction, probesynthesis, and signal amplification in in situ hybridizations.

As used herein, the term “reagent mix” refers to a composition havingall the elements required to perform reverse transcription polymerasechain reaction, or real-time polymerase chain reaction, including butnot limited to primers having specificity for the sequence of thediagnostic target RNA or DNA, respectively, and a polymerase.

As used herein, “primer” refers to an oligonucleotide, synthetic ornaturally occurring, which is capable of acting as a point of initiationof nucleic acid synthesis or replication along a template strand whenplaced under conditions in which the synthesis of a complementary strandis catalyzed by a polymerase. Within the context of reversetranscription, primers are composed of nucleic acids and prime on RNAtemplates. Within the context of PCR, primers are composed of nucleicacids and prime on DNA templates.

As used herein, the term “DNA polymerase” refers to any enzyme thathelps catalyze in the polymerization of deoxyribonucleotides into a DNAstrand. DNA polymerases act to add free nucleotides to the 3′ end of anewly-forming strand, resulting in elongation of the new strand in a5′-3′ direction.

As used herein, “lysis” means perturbation or alteration to a cell wallor viral particle facilitating access to or release of the cellular RNAor DNA. Neither complete disruption nor breakage of the cell wall is anessential requirement for lysis.

As used herein, the term “cycle threshold” or “Ct” refers to the cycleduring thermocycling in which the increase in fluorescence due toproduct formation reaches a significant and detectable level abovebackground signal.

As used herein, the term “direct amplification” refers to a nucleic acidamplification reaction in which the target nucleic acid is amplifiedfrom the sample without prior purification, extraction, orconcentration. It is a relative measure of the concentration of targetin the PCR reaction. Many factors impact the absolute value of Ctbesides the concentration of the target. However, artifacts from thereaction mix or instrument that change the fluorescence measurementsassociated with the Ct calculation will result in template-independentchanges to the Ct value.

As used herein, the term “extraction” refers to any action taken toremove nucleic acids from other (non-nucleic acid) material present inthe sample. Such action includes, but is not limited to, mechanical orchemical lysis, addition of detergent or protease, or precipitation andremoval of non-nucleic acids such as proteins.

As used herein, the term “interfering substance” or “interferent” refersto any substance in a sample that is not a target nucleic acid. Suchinterfering substances include synthetic and biological substances. Suchsynthetic substances include chemicals and pharmaceutical drugs. Suchbiological substances include blood, urine, proteins and otherbiological molecules.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A) is a line graph depicting the results of an H1N1 assay whereinsamples with the FASTSTART® Taq DNA Polymerase buffer (RocheDiagnostics, Indianapolis, Ind.) underwent direct amplificationaccording to the FASTSTART® Taq DNA Polymerase protocol (Roche); (B) isa line graph depicting the results of an H1N1 assay wherein samples withthe GOTAQ® DNA polymerase buffer (Promega) underwent directamplification according to the GOTAQ® DNA polymerase protocol (Promega).

FIGS. 2(A) and (B) are line graphs depicting the effects of two samplestorage buffers: universal transport medium (UTM) (A) and 1× Tris-EDTA(“TE”) (B) were compared using the FASTSTART® Taq DNA Polymeraseprotocol (Roche).

FIG. 3(A) is a line graph depicting the results of an H1N1 assay whereinsamples with the FASTSTART® Taq DNA Polymerase buffer (Roche) underwentamplification according to the FASTSTART® Taq DNA Polymerase protocol(Roche) after nucleic acid extraction; (B) is a line graph depicting theresults of an H1N1 assay wherein samples with the FASTSTART® Taq DNAPolymerase buffer (Roche) underwent direct amplification according tothe Start protocol, without any prior nucleic acid extraction orpurification.

FIGS. 4 (A) and (B) are line graphs demonstrating the effectiveness ofthe GOTAQ® DNA polymerase (Promega) chemistry and cycling conditions fordirect amplification using Influenza A-positive samples.

FIG. 5 is a line graph depicting the sensitivity of direct amplificationassays with added KCl compared with direct amplification assays withoutKCl.

FIG. 6 is a line graph depicting the sensitivity of direct amplificationassays with added surfactant compared with direct amplification assayswithout surfactant.

FIG. 7 is a line graph depicting the sensitivity of direct amplificationassays with pre-heating compared with direct amplification assayswithout pre-heating.

FIG. 8 is a line graph depicting amplification plots from a single bloodsample that was exposed to a different anticoagulant in different tubes(heparin, EDTA, citrate).

FIG. 9 is a line graph depicting the ability of direct amplificationassays to detect samples from buccal swabs.

DETAILED DESCRIPTION

The present invention is directed to diagnostic methods for thedetection of human pathogens including, for example, respiratory virusessuch as influenza A and B viruses and respiratory syncytial viruses(RSV), enterovirus, herpes simplex virus 1 and 2 (HSV-1 and HSV-2,respectively), varicella zoster virus (VZV); and (pathogenic) bacteriasuch as Clostridium difficile using a PCR method that does not involvean extraction or purification step to isolate viral/bacterial (i.e.,target) nucleic acid prior to PCR, and that provides substantiallyequivalent (or better) sensitivity to similar assays using a specificextraction or purification protocol.

More particularly, the present method involves addition of surfactantsto the PCR cocktail to lyse cells or virions, combined certainprocedural steps to increase the availability of target nucleic acids.Samples are then heated to about 50° C. prior to the reversetranscriptase step, and this assists with viral lysis and withinactivation of RNAses. Following the RT step, a PCR reaction isperformed. For bacterial or DNA virus targets, no reverse transcriptaseis required.

Patient samples are added to the reagent mix in a ratio of approximately10-40% patient sample to 60-90% reagent mix, and, optimally, 20-30%patient sample to 70-80% reagent mix. The reagent mix includes apolymerase derived from Thermus aquaticus (e.g., GOTAQ® DNA polymerase;Promega) and an amplification buffer including an ionic detergent (e.g.,GOTAQ® Flexi PCR buffer; Promega). The amplification buffer, which maybe supplied as a 10× buffer, is diluted to about 5×, about 2.5×, orabout 1× concentration for use. The reagent mix further includes KCl(for viral samples only) and MgCl₂, as well as dNTPs. In one formulationencompassed by the present invention, the RT-PCR reagent mixturecontains of: 0.5 μL 5×GOTAQ® Flexi PCR Buffer (Promega), 0.25 μL 25 mMMgCl₂, 0.05 μL 10 mM dNTPs, 0.20 μL 5 U/μL GOTAQ® Flexi DNA polymerase(Promega), and 0.5 μL 10 mM KCl. The patient sample may be heated,either before or after the RT step, and then undergoes PCR.

The reagent mixtures of the present invention allow for the directamplification of nucleic acids from samples without the requirement fornucleic acid extraction or purification prior to amplification. Withoutwishing to be bound by any theory, it is believed that, if required,lysis takes place via a combination of heat and surfactant action.Furthermore, it is believed that the inventive reagent mixturesneutralize amplification inhibitors usually present in RT-PCR reactions,obviating the need for dilution of the specimen; a standard techniqueused in other direct amplification methodologies. The relatively highsalt concentrations may contribute to performance by increasing theoligonucleotide binding efficiencies. However, the reagent mixtures varybased on the type of target nucleic acid.

In one embodiment, the reagent mixture for viral RNA detection includes,at a minimum, a reverse transcriptase, high concentrations of forwardand reverse primers, optimally scorpion primers, MgCl₂, potassiumchloride, dNTPs, 5×GOTAQ® Flexi PCR Buffer (Promega; Cat. No. M891A orM890A) or its equivalent, and a T. aquaticus derivative polymerase suchas 5 U/μl Taq Polymerase (e.g., GOTAQ® Flexi DNA polymerase; PromegaCat. No. M8295). For improved performance, RNAsin may also be added.Additionally, reagent mix for pathogen detection in a spinal fluid orfecal sample also contains BSA.

The reagent mixture for bacterial detection should include, at aminimum, high concentrations of forward and reverse primers, optimallyscorpion primers, MgCl₂, BSA, dNTPs, 5×GOTAQ® Flexi PCR Buffer (Promega;Cat. No. M891A or M890A) or its equivalent, and a T. aquaticusderivative polymerase such as 5 U/μl Taq Polymerase (e.g., GOTAQ® FlexiDNA polymerase; Promega Cat. No. M8295). For improved performance,RNAsin may also be added.

In one embodiment, the assay includes no template control (NTC),positive control, and DNA internal control (IC). The assay can beevaluated based on the Ct values for the controls. In one example, in anassay for detecting C. difficile, if the Ct values for NTC is 0 and ICis ≤40, the control is valid. In another example, in an assay fordetecting C. difficile, if the Ct value for the positive control is 0,the assay is considered invalid. In another example, in an assay fordetecting C. difficile, if the Ct value for C. difficile is ≤40, but ≠0,along with a valid NTC, the assay run is considered valid andacceptable. In another example, in an assay for detecting C. difficile,if the Ct value for C. difficile is =0, and Ct value for IC is ≤40, but≠0, C. difficile is considered not detected. In another example, in anassay for detecting C. difficile, if the Ct value for C. difficile is=0, and Ct for IC=0, the assay is considered invalid.

Source of Viral Particles

Obtaining viral nucleic acid for a detection assay from a sample may beby way of collecting a liquid sample, extracting a solid or semi-solidsample, swabbing a surface, or additional technique. Viral RNA may beassayed directly if the existing concentration adequately providestarget RNA for an RT-PCR reaction. Alternatively, virions may beconcentrated by methods such as centrifugation, binding to a surfacethrough immunoadsorption or other interaction, or filtration.

Source of Bacterial Cells

Obtaining bacterial cells for a detection assay from a sample may be byway of collecting a liquid sample, extracting a solid or semi-solidsample, swabbing a surface, or additional technique. Bacterial cells maybe assayed directly if the existing concentration adequately providestarget RNA for an RT-PCR reaction. Alternatively, bacterial cells may beconcentrated by methods such as centrifugation, binding to a surfacethrough immunoadsorption or other interaction, or filtration. Inaddition, the bacterial cell number may be increased by growing thecells on culture plates or in liquid medium prior to concentration ordirect assay.

Typical bacteria suitable within the context of the invention aregram-negative and gram-positive bacteria including, but not limited to,Listeria, Escherichia, Salmonella, Campylobacter, Clostridium,Helicobacter, Mycobacterium, Staphylococcus, Camplobacter, Enterococcus,Bacillus, Neisseria, Shigella, Streptococcus, Vibrio, Yersinia,Bordetella, Borrelia, and Pseudomonas.

Target Nucleic Acids

RNA types that may be assayed as target nucleic acids include rRNA,mRNA, transfer-RNA (tRNA), or other RNA polynucleotides. Species of rRNAinclude 5S, 16S, and 23S polynucleotides, which may contain one or moresub-sequences characteristic of a group of related bacteria. Thedetection capacity of the characteristic sequence is variable anddepends on the level of relatedness of the virus or bacteria to bedetected by the assay. Other RNA polynucleotides may be used asdiagnostic target RNA so long as they contain unique sub-sequences thatadequately distinguish among bacteria at the desired relatedness level.Examples can be identified from tRNA and mRNA species, as well as fromany RNA produced in a bacterial cell that includes one or morecharacteristic sub-sequence. Primers may be designed by one skilled inthe art to prime the synthesis of a copy DNA using the target RNA astemplate in a reverse transcription reaction. One skilled in the artwill also know how to design pairs of primers for the amplification ofthe unique sub-sequences of the target RNA using the copy DNA astemplate in PCR. It is well known in the art that primers usedsynchronously in PCR should have similar hybridization meltingtemperatures. The diagnostic target RNA within the bacterial cell mustbe made accessible to the RT or RT-PCR reaction composition. After beingcollected, the nucleic acid sample may be directly added to the reactioncomposition, which then undergoes thermocycling.

Although optionally present, a specific lysing agent is preferablyomitted from the reagent mixture because the sufficient release ofviral/bacterial nucleic acids from the sample is obtained without it.Generally, a lysing agent is added prior to contact with the RT, RT-PCR,or PCR reaction composition. The use of lysing agents is well known tothose of skill in the art. Lysing agents include but are not limited tochemicals, enzymes, physical shearing, osmotic agents and hightemperature. By the term “lysis buffer” is meant a buffer that containsat least one lysing agent. Typical enzymatic lysing agents include, butare not limited to, lysozyme, glucolase, zymolose, lyticase, proteinaseK, proteinase E and viral endolysins and exolysins. The viral endolysinsand exolysins are from bacteriophages or prophage bacteria andcombinations of these. Typical viral endolysins include but are notlimited to endolysins from Listeria bacteriophages (A118 and PLYI18),endolysins from bacteriophage PM2, endolysins from the B. subtilisbacteriophage PBSX, endolysins from Lactobacillus prophages Lj928, Lj965and bacteriophage 15 Phiadh, endolysin (Cpl-I) from the Streptococcuspneumoniae bacteriophage Cp-I and the bifunctional peptidoglycan lysinof Streptococcus agalactiae bacteriophage B30. These last two havedifferent bacterial strain specificity. Also contemplated aretwo-component, that is, holin-endolysin, cell 20 lysis genes, holWMY andIysWMY of the Staphylococcus wameri M phage varphiWMY Endolysincombinations of these are also contemplated. For a discussion of virallysis, see especially, Loessner, M J et al. (1995) Applied EnvironmentalMicrobiology I 61: 1150-1152.

Rather than using endolysins, treatment with heat prior to or after theRT step aids in lysis in the present method. Incubation of the sample inthe range of temperature from about 25° C. to less than about 100° C.,and preferably about 50° C. and 75° C., may improve the accessibility ofbacterial RNA as a template for RT or RT-PCR. This heat pretreatment maybe for a time period in the range of about 1 minute to about 60 minutes,with treatments of 1 to 20 minutes being typical, depending on thetemperature of incubation. Heat treatment may include multipleincubations at different temperatures. Heat treatment may be in thepresence or absence of RNase inhibitor as described below. Particularlyuseful treatments are at about 50° C. for about 5 to 20 min in thepresence of RNase inhibitor.

At least one RNAse inhibitor may be added to the virions or bacterialcells. Typically, inhibitors and their concentrations are chosen so asnot to interfere with any of the primer-directed amplification processesand components. RNase inhibitors are known to those of skill in the artand include chemicals such as guanidinium isothiocyanate anddiethyl-pyrocarbonate, protein inhibitors such as Superaseln (Ambion),RNase Block (Stratagene), human placental ribonuclease inhibitor andporcine liver RNase inhibitor (Takara Mirus Bio), anti-nucleaseantibodies such as Anti-RNase (Novagen) and Ribonuclease Inhib III(PanVera), and reagents such as RNAlater (Ambion) and RNA protectBacteria Reagent (Qiagen).

Assay Methods

In the present method, the presence of diagnostic target RNAs is testedby reverse transcription alone or, preferably, by reverse transcriptionand polymerase chain reaction. When used together, reverse transcriptionand polymerase chain reaction may be performed sequentially in twosteps, or together in one step with all reaction composition reagentsbeing added to the sample. Incubation of the sample in the reversetranscription reaction composition allows a DNA copy from the target RNAto be synthesized. The reagent mix includes a primer that hybridizes tothe target RNA to prime the synthesis of the copy DNA. In addition, thereagent mix includes dNTPs, MgCl₂, KCl (in viral samples only), areverse transcriptase and a reverse a transcriptase buffer (in viralsamples only), and, for stool samples, BSA (in bacterial samples only).More than one primer may be included if it is desired to make DNA copiesfrom more than one target RNA. However, no RNase inhibitor is used. Theproduct of the reverse transcription reaction may then be transferred toanother assay tube where PCR is performed according to protocol wellknown in the art. The PCR composition typically includes a pair ofprimers that initiate synthesis of the desired segment of DNA from thereverse transcribed template. In addition, the PCR mix usually comprisesdNTPs, a thermostable DNA polymerase such as Taq polymerase, andpolymerase buffer. More than one pair of primers may be included ifsynthesis of multiple segments of DNA is desired. Also a single newprimer may be added that will amplify a DNA segment with the originalRT-PCR primer as the second primer of the pair. Additional reversetranscriptases that may be used for viral samples include, but are notlimited to, HIV Reverse Transcriptase (Ambion), TRANSCRIPTOR™ ReverseTranscriptase (Roche), and THERMOSCRIPT™ Reverse Transcriptase(Invitrogen). Additional DNA polymerases that may be used include, butare not limited to, Pfu, Vent, and SEQUITHERM™ DNA Polymerase(EPICENTRE™).

Regardless of whether the RT-PCR is carried out as two steps or onestep, the RT step is run first and typically consists of a singletemperature incubation at a temperature of between about 37° C. andabout 70° C. Different temperatures are appropriate for different RTenzymes and different primers, as is known to one skilled in the art.The subsequent PCR reaction typically consists of an initial incubationat about 94° C. to about 96° C. for about 6 to about 15 minutes. Thisstep is used to denature the cDNA and also to activate heat activatedTaq polymerase enzymes. This is then followed by multiple cycles ofamplification of the cDNA target. Three operations are performed duringeach cycle: target denaturation, primer annealing and primer extension.Target denaturation typically occurs at greater than about 90° C. Primerannealing temperature is dictated by the melting temperature of thespecific primers used in the reaction and primer extension is performedat temperatures ranging from about 60° C. to about 72° C. depending onthe thermostable polymerase being used. When primer annealing andextension are performed at the same temperature, this is a twotemperature PCR compared with a three temperature PCR in which each ofthe three steps occur at a different temperature. After theamplification phase is complete, a final extension time is typicallyadded to ensure the synthesis of all amplification products.

Target nucleic acids also include DNA including, for example, DNAderived from bacterial species and DNA viruses. Viral DNA suitable forassessment include both DNA obtained directly from the viral capsid aswell as DNA integrated into the host genome.

Detection of RT and RT-PCR Product

Methods for directly detecting the cDNA product of an RT reaction arewell known to one skilled in the art and make use of labels incorporatedinto or attached to the cDNA product. Signal generating labels that maybe used are well known in the art and include, for example, fluorescentmoieties, chemiluminescent moieties, particles, enzymes, radioactivetags, or light emitting moieties or molecules. Fluorescent moieties areparticularly useful, especially fluorescent dyes capable of attaching tonucleic acids and emitting a fluorescent signal. A variety of dyes areknown in the art such as fluorescein, Texas Red, and rhodamine.Particularly useful are the mono reactive dyes Cy3 and Cy5, bothavailable commercially (from, for example, Amersham Pharmacia Biotech,Arlington Heights, Ill.). A more sensitive way to specifically detectthe labeled DNA is to hybridize the products against target DNA sequencemolecules that are immobilized in a matrix, such as a nylon membrane ora glass slide. The signals after hybridization can then be scanned witha laser scanner with appropriate filtering to detect the specific dyeused. This is well known in the art, especially in DNA microarraytechnology. A label may be incorporated into the cDNA during itssynthesis in the RT reaction, or it may be attached to the cDNA productafter its synthesis. For example, the RT reaction can be carried outwith labeled primers. One type of labeled primer has attached particleshaving a large number of signal generating molecules. Reversetranscription using a labeled nucleotide, such as dye-labeled UTP and/orCTP, incorporates a label into the transcribed nucleic acids.Alternatively, a post-synthesis coupling reaction can be used to detectthe cDNA products. Attaching labels to nucleic acids is well known tothose of skill in the art and may be done by, for example, end-labelingwith, e.g. a labeled RNA or by treatment of the nucleic acid with kinaseand subsequent attachment of a nucleic acid linker joining the samplenucleic acid to the label, e.g., a fluorophore. In another labelingmethod, the DNA products from the RT reaction are amplified by couplingto an in vitro transcription reaction. For example, the T7 promoterregion is incorporated into the primer used for the RT reaction. A T7 invitro transcription kit can then be used to generate a large amount ofRNA to increase the detection sensitivity. The T7 in vitrotranscriptional kit can be purchased from Ambion (2130 Woodward, Austin,Tex.) or other commercial sources.

RT-PCR Detection

Methods for RT-PCR product detection include gel electrophoresisseparation and ethidium bromide staining, or detection of anincorporated fluorescent label or radiolabel in the product. Methodsthat do not require a separation step prior to detection of theamplified product may also be used. These methods are commonly referredto as Real-Time PCR or homogeneous detection. Most real time methodsdetect amplified product formation by monitoring changes in fluorescenceduring thermocycling. These methods include but are not limited to:TAQMAN® dual labeled probes (Applied Biosystems, Foster City, Calif.94404), Molecular Beacons (Tyagi S and Kramer F R (1996) Nat Biotechnol14:303-308), and SYBR® Green dye (Molecular Probes, Inc Eugene, Oreg.97402-0469). Some of these same homogeneous methods can be used for endpoint detection of amplified products as well. An example of this typeof method is SYBR® Green dye (Molecular Probes) dissociation curveanalysis. In dissociation curve analysis a final slow ramp intemperature, generally about 60° C. to 90° C., combined withfluorescence monitoring can detect the melting point and thereby thepresence of an amplified product (Ririe et al. (1997) Anal. Biochem.245: 154-60).

Assay Sensitivity

The sensitivity of the direct amplification assays can be increased byadding one or more sensitivity-increasing components to the buffer usedin the assays. Such components include, but are not limited to, KCl, asurfactant and albumin. In some embodiments, the albumin is bovine serumalbumin. In some embodiments, the surfactant is a cationic surfactant.The sensitivity of the direct amplification assays also can be increasedby providing additional heating to the assays, such as pre-heating asample before the reagents are added. In some embodiments, thesensitivity can be increased by a combination of thesensitivity-increasing components and additional heating.

EXAMPLES

The present methods, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentmethods and kits.

Example 1: Universal Master Mix

Except as otherwise noted, the 2.5× Universal Master Mix (UMM) isprepared in the following proportions. The table below provides thevolume of reagents suitable to prepare 15 ml of UMM, however, anysuitable volume may be prepared according to need.

Reagent Volume per 15 ml 5X GOTAQ ® Flexi PCR Buffer (Promega; 5.0 mlCat. No. M891A or M890A) 25 mM MgCl₂ 2.5 ml 10 mM dNTPs (10 mM for eachof dATP, 0.5 ml dGTP, dCTP, and dTTP) 5 U/μl Taq Polymerase (e.g.,GOTAQ ® 2.0 ml FlexiDNA Polymerase; Promega Cat. No. M8295) 10 mM KCl5.0 ml

The GOTAQ® Flexi PCR buffer (Promega) and equivalents contain ionicdetergents and lack magnesium.

Example 2: Effect of KCl on Direct Amplification of Respiratory VirusNucleic Acids

Contrived swab specimens containing influenza B virus were prepared byadding cultured influenza B virus (“Flu B”) (Great Lakes strain) toviral transport medium samples, along with an internal control nucleicacid. The Ct values for the Flu B and control nucleic acids weredetermined in the presence and absence of 25 mM KCl, using the UniversalMaster Mix of Example 1, with the omission of KCl from the UMM, andfurther containing either 1 or 2 μl of reverse transcriptase (IMPROM-II™Reverse Transcriptase, Promega Cat. No. A3800). Serial dilutions oftemplate copies of Flu B virus were assessed at TCID₅₀/ml of 158 and39.5. The RT-PCR reaction was run as follows:

Stage 1: 75° C. for 3 min (once)

Stage 2: 47° C. for 10 min (once)

Stage 3: 97° C. for 2 min (once)

Stage 4: 102° C. for 1 sec. followed by 60° C. for 20 sec. for datacollection (repeated 45 times)

The threshold for Ct determination was 50,000. The fluorophores for theFlu B and internal control probes were JOE™ fluorophores (LifeTechnologies) and Q670, respectively. All assays were run in duplicateand the results averaged.

TABLE 1 Effect of KCl on Direct RT-PCR From Serum (Flu B TCID₅₀/ml =158) Reverse Flu B Flu B Internal Control 25 mM KCl Transcriptase CtAvg. Ct Ct + 1 μl 34.2 34.0 38.4 + 1 μl 33.8 36.2 + 2 μl 33.7 33.737.8 + 2 μl 33.7 38.4 — 1 μl 34.0 34.4 34.6 — 1 μl 34.8 34.6 — 2 μl 33.633.9 36.2 — 2 μl 34.2 34.9

TABLE 2 Effect of KCl on Direct RT-PCR From Serum (Flu B TCID₅₀/ml =39.5) Reverse Flu B Flu B Internal Control 25 mM KCl Transcriptase CtAvg. Ct Ct + 1 μl 35.9 36.3 37.6 + 1 μl 36.7 40.2 + 2 μl 35.4 35.636.2 + 2 μl 35.7 39.1 — 1 μl 35.5 37.6 41.1 — 1 μl 39.6 35.6 — 2 μl 35.736.1 35.8 — 2 μl 36.4 36.3

The results in Tables 1 and 2 demonstrate that the presence of KClenhances the sensitivity of a direct RT-PCR amplification assay forrespiratory viruses (Flu B) in serum at low viral concentrations. Theseresults further indicate that the presence of KCl mitigates or negatesthe necessity for concentrating and/or purifying virus from serum priorto analysis by RT-PCR.

Example 3: Direct Amplification of Respiratory Virus Nucleic Acids fromClinical Samples

Various clinical samples (buccal swab and cerebrospinal fluid) wereassessed for the presence of HSV-1 and/or HSV-2. The Universal MM fromExample 1 was used, with the noted modifications to MgCl₂ and KCl.

The buccal swab RT-PCR amplification master mix was:

Component Concentration 2.5X Universal MM 1x Scorpion Forward Primer 600nM Reverse Primer 600 nM MgCl₂ 5 mM Potassium Chloride 40 mM

The CSF RT-PCR amplification master mix was:

Component Concentration 2.5X Universal MM 1x Scorpion Forward Primer 600nm Reverse Primer 600 nm MgCl₂ 2.5 mM 100X BSA (10 mg/ml) 0.1-0.5 mg/mlPotassium Chloride 40 mM

Clinical samples were analyzed using both the direct RT-PCRamplification protocol and an RT-PCR amplification protocol that used aninitial nucleic acid extraction protocol. Nucleic acids were extractedusing a Roche MagNA Pure LC instrument (Roche), and the correspondingTotal Nucleic Acid Isolation Kit. A total of 200 μl of sample wasextracted, and the nucleic acid was eluted in 50 μl.

For each assay, 10 μl of sample was added to 40 μl of the Master mixdescribed in this Example. The RT-PCR reaction was run as follows:

Stage 1: 75° C. for 3 min (once)

Stage 2: 47° C. for 10 min (once)

Stage 3: 97° C. for 2 min (once)

Stage 4: 102° C. for 1 sec. followed by 60° C. for 10 sec. for datacollection (repeated 50 times)

The results are as follows:

TABLE 3 Multiplex Amplification Assessment of Multiple RespiratoryViruses in Clinical Samples Ct Value With Ct Value With DirectAmplification VBS Sample Extraction Internal ID# Type HSV-1 HSV-2 HSV-1HSV-2 Control 42731 Swab N/A 28.2 ND 29.2 33.4 72734 Swab N/A 24.1 ND29.1 34.1 42735 Swab N/A 17.5 ND 18.2 41.9 42933 Swab 0.0 31.5 ND 32.835.0 42944 CSF 0.0 0.0 ND ND 35.3 42946 CSF 0.0 0.0 ND ND 35.3 42947 CSF0.0 0.0 ND ND 36.2 041378 Swab 20.7 ND 20.1 ND 33.8 041379 Swab 26.6 ND25.2 ND 32.8 42846 Swab 0.0 0.0 ND ND 35.0 42847 Swab 0.0 0.0 42.7 ND34.4 42848 Swab 0.0 0.0 ND ND 33.9 42827 Swab 34.2 0.0 34.4 ND 33.242839 Swab 0.0 33.4 ND 35.8 34.5 42952 CSF 34.5 0.0 33.8 ND 34.1 42955CSF 30.2 0.0 34.6 ND 44.6 42963 CSF 32.9 0.0 34.0 ND 35.8 42984 CSF 0.037.4 ND 40.0 ND 43009 CSF 0.0 45.2 ND 40.5 37.7 42975 CSF 0.0 37.6 ND39.0 38.7 42995 CSF 0.0 34.1 ND 35.1 35.8 43010 CSF 0.0 37.9 ND 38.437.9 43001 CSF 0.0 32.3 ND 33.6 37.8 42957 CSF 27.3 0.0 32.0 ND 35.342959 CSF 30.1 0.0 30.1 ND 35.1 42961 CSF 31.3 0.0 32.0 43.1 34.8 39929Swab 0.0 38.1 ND 24.1 ND 39984 Swab 39.8 0.0 37.6 ND 36.3 39713 Swab38.5 0.0 30.8 43.3 36.6 39724 Swab 26.0 0.0 28.5 ND 35.6

These data demonstrate that for HSV infected buccal or CSF samples, thedirect amplification method described above provides comparable resultsin a multiplex RT-PCR amplification assay relative to an RT-PCR protocolthat performs a nucleic acid extraction and concentration prior atanalysis.

Example 4: Effect of the RNAse Inhibitors on Direct Nucleic AcidAmplification Assays

A control virus was spiked into viral transport media to create asynthetic sample for analysis using the direct amplification RT-PCRassay described above. For each assay, 10 μl of sample was added to 40μl of the UMM described in Example 1, in the presence or absence of 1 μlof an RNAse Inhibitor (“RNAsin”) (Promega Cat. No. N261B). The RT-PCRreaction was run as follows:

Stage 1: 50° C. for 10 min (once)

Stage 2: 97° C. for 2 min (once)

Stage 3: 102° C. for 1 sec. followed by 58° C. for 20 sec. for datacollection (repeated 50 times)

In the absence of the RNAsin, the Ct could not be determined suggestingthat the viral RNA was degraded prior to RT. The average Ct in thepresence of RNAsin was 32.5 from assays run in duplicate. These resultsdemonstrate that the presence of RNAsin improves the sensitivity of thedirect RT-PCR amplification method.

Example 5: Effect of Sample Pre-Heating on Direct Nucleic AcidAmplification Assays

Viral transport media was spiked with a combination of influenza A,influenza B, and RSV viruses at approximately 5,000 virus copies/ml toform “FABR” synthetic samples, or with Influenza B virus (10⁻⁴) alone.These samples were assessed by direct amplification using the UniversalMaster Mix of Example 1 with the addition of 1 μl Improm II reversetranscriptase and 0.25 μl RNAsin. MS2 phage was added as an internalcontrol. Experimental samples were pre-heated for 3 min at 75° C. and 10μl of sample was added to 40 μl of Universal Master Mix for each assay.The RT-PCR reaction was run as follows:

Stage 1: 50° C. for 10 min (once)

Stage 2: 97° C. for 2 min (once)

Stage 3: 102° C. for 1 sec. followed by 58° C. for 20 sec. for datacollection (repeated 50 times)

The results are as follows:

TABLE 4 Multiplex Amplification Assessment of Multiple RespiratoryViruses in Clinical Samples With Sample Pre-heating Calculated Ct ValueFlu A Flu B RSV Internal Control FABR w/pre-heat 33.5 34.0 33.8 34.6 FluB w/pre-heat ND 32.3 ND 33.8 FABR w/out pre- 33.3 ND 37.4 33.8 heat FluB w/out pre- ND ND ND 33.9 heat

Next, control cerebral spinal fluid samples were spiked with HSV-1 virus(TCID₅₀/ml=2.14) and control virus were assessed with and without samplepre-heating in the absence of RNAsin. Specificity of the HSV-1 detectionmethodology was confirmed by simultaneously assessing the samples forthe presence of HSV-2 nucleic acid. HSV-2 was not detected in anysample. The results are as follows:

TABLE 5 Direct Amplification Assessment of HSV in Clinical Samples WithSample Pre-heating No Pre-heating With Pre-heating HSV-1 I.C. HSV-1 I.C.Sample #1 38.5 31.3 36.6 29.9 Sample #2 38.3 31.0 35.4 29.7 Sample #3 ND30.9 35.4 29.7 Sample #4 39.6 31.0 35.6 29.8 Sample #5 41.1 31.1 36.229.8 Sample #6 38.1 30.9 35.5 29.9 Sample #7 37.6 31.2 35.6 29.6 Sample#8 40.0 31.2 35.5 29.8 Average 39.0 31.1 35.7 29.8

These results demonstrate that briefly pre-heating the samples prior todirect RT-PCR amplification and assessment enhances the sensitivity ofviral detection in most cases (i.e., at least for FluB, RSV, and HSV-1)and does not negatively influence the sensitivity for others (i.e.,FluA). Furthermore, sample pre-heating reduces or eliminates the needfor the inclusion of an RNAse inhibitor.

Example 6A: Stool Sample Protocol with BSA

A human stool sample is obtained from a patient using standard clinicalmethodology. Samples are maintained at 2-25° C. for transport andshort-term storage and subjected to not more than one freeze/thaw cycleprior to use. For assessment, a flocked swab is dipped into athoroughly-mixed stool specimen and the excess stools is removed bypressing the swab against the side of the specimen container. The swabis then swirled in 1 ml of Tris-EDTA (TE) buffer and discarded. Thesample is heated at 97° C. for 10 minutes. The sample is then diluted1:4 using the UMM of Example 1 (i.e., 2 μl of sample is added to 8 μl ofUMM) with the UMM modifications noted below. Optionally, 2 μl of asolution containing a positive internal control nucleic acid may beadded to 8 μl of UMM, which contains 4 μl of the UMM, along with 0.35mg/ml BSA, 600 nM each of the forward and reverse primers, 300 nM eachof internal control primers, and 0.5 μl of internal control DNA). The C.difficile target primer was labeled with a FAM™ fluorophores (LifeTechnologies, Carlsbad, Calif.), and the internal control target waslabeled with a Quasar670 fluorophore. Thermocycling began with aninitial denaturation step at 97° C. for 2 minutes, followed by 40 cyclesof 97° C. for 10 seconds, and 60° C. for 30 seconds. Real-time PCR wasperformed for 40 cycles and the amplification curves was determinedusing fluorescently-labeled probes specific for the target nucleic acid,which in this experiment was the C. difficile TCD-B gene.

Component Concentration 2.5X Universal MM 1x Scorpion Forward Primer 600nM Reverse Primer 600 nM MgCl₂ 5 mM 100X BSA (10 mg/ml) 0.35 mg/ml

Example 6B: Stool Sample Protocol without BSA

In order to determine whether the addition of BSA is likely to decreaseinhibition of detection resulting from stool material, the RT-PCR assaywas performed according to the protocol used for testing samples withBSA using Clostridium difficile DNA as a target both in the presence andabsence of BSA. The stool samples were prepared according to theformulation shown in Example 6A, although the BSA-negative samplesomitted the 100×BSA. The MagnaPure system (Roche) was used for nucleicacid purification. The PCR reaction was performed as described above,with the BSA-negative PCR formulation plated in wells 1-20, and theBSA-positive PCR formulation was plated in wells 21-40.

The results, which are shown in Table 6, indicate that BSA additiondecreases inhibition of gram-positive anaerobic bacterial nucleic aciddetection from a stool sample.

TABLE 6 Well C. Diff I.C. regular reaction mix 1 32.8 28.2 2 31.2 27.7 330.4 27.4 4 31.3 28.2 5 31.0 28.1 6 35.5 27.3 7 35.8 27.4 8 0 0 9 36.727.0 10 36.0 27.2 11 30.4 27.1 12 30.7 27.5 13 0 0 14 30.6 27.4 15 0 016 34.5 30.7 17 36.8 26.8 18 0 29.0 19 0 27.5 20 31.3 27.9 BSA reactionmix 21 33.3 27.7 22 31.4 28.1 23 30.5 27.7 24 31.2 28.0 25 30.8 28.4 2634.6 27.1 27 36.4 27.4 28 31.3 27.7 29 43.1 27.2 30 35.6 27.1 31 30.827.3 32 30.6 27.5 33 0 29.3 34 30.8 27.4 35 0 30.9 36 34.4 29.8 37 35.827.2 38 40.1 27.6 39 0 26.7 40 31.4 27.8

Example 7: Direct RT-PCR Amplification is Buffer-dependent

Samples of Influenza A, and an internal control were assessed usingdirect amplification to determine whether the enzymes used in theUniversal Master Mix as defined in Example 1 are unique in their abilityto facilitate a direct detection assay. A hybrid primer concentrationwas used consisting of 600 nM influenza A scorpion/primer (directed tothe matrix gene), 500 nM swine H1 scorpion/primer (directed to thehemagglutinin gene), and 150 nM armored RNA internal control (IC)scorpion primer specific for MS2 phage. The reaction mix was preparedusing two distinct enzymes and buffer systems: GO TAQ FLEXI® DNAPolymerase, along with its accompanying 5×PCR Buffer (Promega; Cat. No.M891A or M890A) as a component of universal MM, and FASTSTART® HighFidelity PCR System (Roche), with its accompanying 10× Buffer, as acomponent of RNA MM, in concentrations as follows:

TABLE 7 Reaction Mix Components and Concentrations FASTSTART ® GOTAQ ®RNA MM 4.0 μL 2.5X universal MM 4.0 μL Improm II RT 0.5 μL Improm II RT0.5 μL RNAse inhibitor 0.2 μL RNAse inhibitor 0.2 μL 20X H1N1 primer mix0.5 μL 20X H1N1 primer mix 0.5 μL 1:100000 MS2 phage 0.5 μL 1:100000 MS2phage 0.5 μL Water 2.3 μL Water 2.3 μL Sample 2.0 μL Sample 2.0 μL

The samples consisted of influenza viruses that were spiked into viraltransport media. A total of 2 μl of specimen was added to 8 μl of eachreaction mix listed above. Each sample was amplified directly withoutpre-extraction.

Thermocycling was then performed, and amplification curves aredetermined using fluorescently-labeled probes specific for the targetnucleic acids. InfA M gene probes, H1N1-specific HA gene probes, and ICprobes were labeled with FAM™ fluorophore (Life Technologies), CFR610,and Q670, respectively. Two cycling protocols were then used for RT-PCR(ICy/Universal 96-well disc; 3M). The GOTAQ® DNA Polymerase (Promega)cycling protocol and the FASTSTART® Taq DNA Polymerase (Roche) cyclingprotocol assays were first performed with GOTAQ® DNA Polymerase(Promega) cycling protocol, then both were performed with FASTSTART® TaqDNA Polymerase (Roche) cycling protocol. The FASTSTART® Taq DNAPolymerase (Roche) cycling protocol was as follows:

Stage 1: 47° C. for 15 min (once)

Stage 2: 97° C. for 10 min (once)

Stage 3: 97° C. for 15 sec. followed by 60° C. for 30 sec. (repeated 40times)

The GOTAQ® DNA Polymerase (Promega) cycling protocol was as follows:

Stage 1: 47° C. for 10 min (once)

Stage 2: 97° C. for 2 min (once)

Stage 3: 97° C. for 5 sec. followed by 58° C. for 30 sec. (repeated 40times)

The results are shown in FIGS. 1(A) and (B) which represent samples withFastStart buffer run using FastStart cycling protocol, and with GOTAQ®DNA Polymerase buffer (Promega) run using GOTAQ® DNA Polymerase(Promega) cycling protocol. No amplification was observed withFASTSTART® Taq DNA Polymerase (Roche) cycling conditions and buffersystem, whereas robust amplification was observed using the GOTAQ® DNAPolymerase (Promega) cycling conditions and buffer system.

The effect of sample storage buffer was also compared. Specifically,H1N1 nucleic acid amplification from samples stored in universaltransport medium (UTM) or 1× Tris-EDTA (“TE”) was compared using theFASTSTART® Taq DNA Polymerase protocol (Roche). As shown in FIG. 2, nosignificant amplification was observed in the UTM samples, whereas theTE samples yielded robust amplification curves.

The effect of nucleic acid extraction on the FASTSTART® Taq DNAPolymerase (Roche) buffer chemistry and cycling conditions wasinvestigated. As shown in FIG. 3, it was confirmed that the FASTSTART®Taq DNA Polymerase (Roche) protocol failed to amplify H1N1 nucleic acidsdirectly from clinical samples (FIG. 3B). However, robust amplificationwas observed for samples in which the nucleic acids were extracted priorto the amplification reaction (FIG. 3A). These results demonstrate thatnot all RT-PCR amplification conditions may be applied to directamplification/detection systems and that the GOTAQ® DNA Polymerase(Promega) chemistry is particularly suited for this assay format.

The effectiveness of the GOTAQ® DNA Polymerase (Promega) bufferchemistry and cycling conditions for direct amplification was confirmedusing Influenza A-positive patient samples (including 2009 pandemic H1N1positive samples). Amplification was performed using the followingparameters:

PCR Reaction Setup 2.5X universal MM 4.0 μL Improm II RT 0.5 μL RNAseinhibitor 0.2 μL 20X H1N1 primer mix 0.5 μL 1:100000 MS2 phage 0.5 μLWater 2.3 μL Sample 2.0 μL

Cycling Conditions

Stage 1: 47° C. for 10 min (once)

Stage 2: 97° C. for 2 min (once)

Stage 3: 97° C. for 5 sec. followed by 58° C. for 30 sec. (repeated 40times)

The results shown in FIG. 4A are from samples containing non-pandemicinfluenza A virus, and amplification of the FAM™ fluorophore (LifeTechnologies) target indicates detection of influenza A, but not ofpandemic H1N1 influenza A. FIG. 4B shows amplification of pandemic H1N1samples, and demonstrates amplification of the influenza A target, alongwith the H1N1-specific target.

Example 8: Detection of Flu a, Flu B and RSV by Direct Nucleic AcidAmplification Assays and Comparison with Methods Using Nucleic AcidExtraction

Nucleic acid from the clinical specimens (swabs) and control sampleswere amplified using the direct nucleic acid amplification assays andthe results were compared with amplification results using methodsinvolving nucleic acid extraction. The sequences of the amplificationprimers are shown in the table below.

Name Sequence Univ Flu A 5′ BHQ-1-ACGCTCACCGTGCCCAGTGAGCG- ScorpionT(FAM)-Spacer 18-GGCATTTTGGACAAAGCGTC TA 3′ (SEQ ID NOS: 1-2)Univ Flu A  5′ TCTTGTCACCTCTGACTAAGGGGAT 3′ Rev Primer (SEQ ID NO: 3)Flu B  5′ JOE-C6-CCGCGG-I-ATTGCAAAGGATGTAATG ScorpionGAAGTGCCGCGG-BHQ-1-Spacer 18- GAGCTGAATTTCCCAT-I-GAGCT 3′(SEQ ID NOS: 4-5) Flu B Rev  5′ AGCTGCAAAGCAACATTGGAG 3′ Primer(SEQ ID NO: 6) RSV A/B  5′ CAL Fluor Red 610-ACGCGCTTCACGAAGG ScorpionCTCCACATACACAGCGCGT-BHQ-2-Spacer 18- TTTTCTAGGACATTGTAYTGAACAG 3′(SEQ ID NOS: 7-8) RSV A/B Rev 5′ GCAAATATGGAAACATACGTGAACAA 3′ Primer(SEQ ID NO: 9) RNA IC  5′ Quasar 670-ACGCGCTTGGGGCGACAGTCAC ScorpionGTCGCGCGT-BHQ-2-Spacer 18-CTCGTCGACA ATGGCGGAA 3′ (SEQ ID NOS: 10-11)RNA IC Rev 5′ TTCAGCGACCCCGTTAGC 3′ Primer (SEQ ID NO: 12)

Clinical specimens and control samples were heated at 65° C. to 70° C.for 5 min. The reaction mixture was prepared as follows:

Reaction Mixture

Reaction Component Volume (μL) 2.5X master mix 4.0 Reverse transcriptase0.5 RNase inhibitor 0.2 50X flu A primer pair 0.2 50X flu B primer pair0.2 50X RSV primer pair 0.2 50X internal control primer pair 0.2Internal control RNA (heated) 0.5 Nuclease-free water 2.0 Reaction Mix8.0 Specimen (heated) 2.0 Total reaction volume 10.0Thermocycling was then performed using the following cycling parameter.Cycling Parameters

Step Time (sec) Temp (° C.) Repeat cDNA synthesis 600 47 1 Initialheating 120 97 1 Denaturation 5 97 40 Anneal/extension 30 58

The amplification results using the direct nucleic acid amplificationassays were compared with the amplification results obtained usingmethods involving nucleic acid extraction as shown below.

Clinical Specimens (Swabs) Tested

Previous result Number of specimens Flu A positive 35 Flu B positive 91RSV positive 47 Negatives 19 Total 193

There was 100% concordance between the results obtained using the directnucleic acid amplification assays and the results obtained using methodsinvolving nucleic acid extraction as shown above.

Example 9: Detection of HSV-1, HSV-2, and VZV by Direct Nucleic AcidAmplification Assays and Comparison with Methods Using Nucleic AcidExtraction

Nucleic acids from the clinical specimens as well as contrived sampleswere amplified using the direct nucleic acid amplification assays andthe results were compared with amplification results using methodsinvolving nucleic acid extraction. The reaction mixture was prepared asfollows:

Reaction Mixture

Reaction component Volume (μL) 2.5X master mix 4.0 25X HSV1 primer pair0.4 25X HSV2 primer pair 0.4 50X VZV primer pair 0.2 50X Internalcontrol primer pair 0.2 Internal control DNA 0.2 Nuclease-free Water 0.6Reaction mix 6.0 Sample 4.0 Total reaction volume 10.0

The sequences of the amplification primers are shown in the table below.

Sequences

Sequence Name Sequence HSV-1 ScorpionCFR610-AGCGGCCCGGGTGCCCGGCCAGCCGCT- BHQ-2-Spacer 18-GAGGACGAGCTGGCCTTTC(SEQ ID NOS: 13-14) HSV-2 Scorpion BHQ1-ACGCGCTTCCGGGCGTTCCGCGAGCGCG-T(FAM)-Spacer 18-GAGGACGAGCTGGCCTTTC (SEQ ID NOS: 15-16) HSV-1&2 primerGGTGGTGGACAGGTCGTAGAG (SEQ ID NO: 17) VZV ScorpionJOE-C6-ACGCGGCTTCTGTTGTTTCGACCGCGT- BHQ-1-Spacer 18-CCCCGCTTTAACACATTCCA(SEQ ID NO: 18-19) VZV primer GCAGTTGCAAACCGGGAT (SEQ ID NO: 20)Thermocycling was performed using the following cycling parameter.Cycling Parameters

Step Time (sec) Temp (° C.) Repeat Initial heating 120 97 1 Denaturation10 97 40 Anneal/extension 30 60

The amplification results using the direct nucleic acid amplificationassays were compared with the amplification results obtained usingmethods involving nucleic acid extraction as shown below.

VZV

A total of 32 out of 32 specimens (13 swabs, 2 vitreous fluid, 17 CSF)were detected as positive for VZV using amplification methods involvingnucleic acid extraction, while 31 out of 32 specimens detected aspositive by the direct amplification method. The CSF sample that wastested negative, was detected with Ct 37.1 when tested with nucleaseinhibitor.

HSV-1 and HSV-2

Clinical Specimens (Swabs):

Two HSV-1 samples that were detected as positive using amplificationmethods involving nucleic acid extraction were also tested as positiveusing the direct amplification method. Similarly, two HSV-2 samples thatwere detected as positive using amplification methods involving nucleicacid extraction were also tested as positive using the directamplification method.

Contrived Samples:

The detection results using the contrived samples in the directamplification versus an extraction of nucleic acid prior toamplification are shown below for HSV-1 and HSV-2.

HSV-2

HSV2 Universal Direct Extracted method (TCID₅₀*/mL) detected/totaldetected/total 2.8 × 10³ 1/1 1/1 1.4 × 10² 1/1 1/1 1.4 × 10¹ 2/2 2/2 1.4× 10⁰ 2/2 2/2  1.4 × 10⁻¹ 2/2 2/2 *TCID₅₀: 50% tissue culture infectivedoseHSV-1

HSV1 Universal Direct Extracted method (TCID₅₀*/mL) detected/totaldetected/total 1.8 × 10³ 1/1 1/1 0.9 × 10³ 2/2 2/2 1.8 × 10² 2/2 2/2 0.9× 10² 2/2 2/2 1.8 × 10¹ 1/2 2/2 *TCID₅₀: 50% tissue culture infectivedose

Thus, the results using the direct amplification method and the methodusing nucleic acid extraction are comparable for detecting HSV-1 andHSV-2.

Example 10: Detection of Enterovirus by Direct Nucleic AcidAmplification Assays and Comparison with Methods Using Nucleic AcidExtraction

Nucleic acids from the samples were amplified using the direct nucleicacid amplification assays and the results were compared withamplification results using methods involving nucleic acid extraction.The reaction mixture was prepared as follows:

Reaction Mixture

Reaction Component Volume (μL) 2.5X master mix 4.0 Reverse transcriptase0.5 RNase inhibitor 0.1 50X entero virus primer pair 0.2 50X internalcontrol primer pair 0.2 Internal control RNA 0.1 Reaction Mix 5.0 Sample5.0 Total reaction volume 10.0

The sequences of the amplification primers are shown in the table below.

Sequences

Sequence Name Sequence Enterovirus BHQ1-AGGCCACACGGACACCCAAAGTAGTCGGTGGScorpion CC-T(FAM)-Spacer 18-CCCCTGAATGCGGCTA ATC (SEQ ID NOS: 21-22)Enterovirus CAATTGTCACCATAAGCAGCCA primer (SEQ ID NO: 23)

Thermocycling was performed using the following cycling parameter.

Cycling Parameters

Step Time (sec) Temp (° C.) Repeat cDNA synthesis 600 47 1 Initialheating 120 97 1 Denaturation 5 97 45 Anneal/extension 30 58

The amplification results using the direct nucleic acid amplificationassays were compared with the amplification results obtained usingmethods involving nucleic acid extraction as shown below.

32 samples testing negative by amplification methods involving nucleicacid extraction were also negative by the direct amplification method.16 samples testing positive by amplification methods involving nucleicacid extraction were also positive by the direct amplification method.

The detection results using the contrived samples in the directamplification versus an extraction of nucleic acid prior toamplification are shown below.

Direct Method using Nucleic Enterovirus Amplification Acid Extractionmethod (TCID₅₀/mL) detected/total detected/total 9.0 × 10⁴ 1/1 1/1 9.0 ×10³ 1/1 1/1 9.0 × 10² 2/2 2/2 9.0 × 10¹ 2/2 2/2 9.0 × 10⁰ 2/2 2/2 4.5 ×10⁰ 4/4 4/4 1.8 × 10⁰ 4/4 4/4 TCID₅₀: 50% tissue culture infective dose

Thus, the results using the direct amplification method and the methodusing nucleic acid extraction are comparable for detecting Enterovirus.

Example 11: Detection of Clostridium difficile by Direct Nucleic AcidAmplification Assays and Comparison with Methods Using Nucleic AcidExtraction

Nucleic acids from the samples were amplified using the direct nucleicacid amplification assays and the results were compared withamplification results using methods involving nucleic acid extraction.

Stool specimens were obtained from different individuals. Flocked swabwas dipped into the stool specimen. Excess stool specimen was removed.The swab was placed in 1 ml of TE buffer, swirled, and the swab wasdiscarded. The samples were heated at 97° for 10 min in a heating block.

The PCR master mix was prepared as follows:

PCR Mix

Component Concentration 2.5X Universal MM 1x Scorpion Forward Primer 600nM Reverse Primer 600 nM MgCl₂ 5 mM 100X BSA (10 mg/ml) 0.35 mg/ml

Two microliters of the heated sample was added to eight microliters ofthe master mix and the PCR was carried out using the following cyclingparameters:

Step Cycles Temp (° C.) Time 1 1 97 2 min 2 40 97 10 sec 60 30 sec

The primers target toxin B region of C. difficile. The sequences of theamplification primers and the amplicon are shown below.

C difficile Scorpion primer: 5′d BHQ-1-AGGCAGCTCACCATCAATAATAACTGAACCAGTTGCTGCC-T(FAM)-Spacer 18-GGTTAGATTTAGATGAAAAGAGATATTATTT TA 3′(SEQ ID NOS: 24-25) C difficile Reverse primer: 5′d ACTAATCACTAATTGAGCTGTATCAGGA 3′ (SEQ ID NO: 26) C. difficile amplicon:Ggttagatttagatgaaaagagatattattttacagatgaatatattgcagcaactggttcagttattattgatggtgaggagtattattttgatcctgatacagctcaattagtgattagt (SEQ ID NO: 27)

Fluorescent signal from C. difficile Scorpion primers was detected at495 nm and the signal from internal control was detected at 644 nm.

The amplification results using the direct nucleic acid amplificationassays were compared with the results obtained using amplificationmethods involving nucleic acid extraction as shown below.

Methods involving nucleic acid extraction % Positive Negative TotalAgreement Direct Posi- 109 7 116 99.1% amplification tive (109/110)method Nega- 1 72 73 91.1% tive (72/79) Total 110 79 189

Thus, 99% of the samples identified positive by amplification methodsinvolving nucleic acid extraction were also identified positive by thedirect amplification assay, and 91% of the samples identified negativeby amplification methods involving nucleic acid extraction also wereidentified negative by the direct amplification assay.

The Limit of Detection (LoD) was determined using a panel consisting ofcontrived samples in stool-TE buffer matrix, spiked with C. difficilebacterial stock. The panel included negatives (unspiked matrix) andsamples of varying concentrations around the approximate LoD (obtainedin an earlier phase of testing). Results (positive/negative) of twentyfour (24) replicates from three (3) distinct preparations and PCR runs(eight replicates/run) at each level were analyzed with Probit Analysisto determine the lowest concentration which could accurately be detectedwith 95% probability. The limit of detection is 0.04 cfu/reaction.

Reproducibility of the Assay

Reproducibility study was performed using contrived samples in stool-TEbuffer matrix, spiked with C. difficile bacterial stock. The panelincluded a negative (unspiked matrix), a low positive (approximately 2to 4 times LOD), and a medium positive (approximately 8 to 10 times LOD)samples. The Reproducibility study was performed using two integratedcycler instruments for five days (not consecutive days). Each day, tworuns were performed on each instrument. Each run included fourreplicates of each panel member and positive control (PC) and onereplicate of no template control (NTC). The panel and PC were assayedwith four replicates, and NTC, in singlicate in each run of theIntegrated Cycler instrument. One lot of direct amplification assays wasused to run the panel over a period of five days (not consecutive days)at two runs per day per instrument. There was a minimum of twoinstruments with at least one operator per instrument. The summary ofthe reproducibility of the results is shown below.

Quantitative Summary of Reproducibility Inter-Instrument Inter-DayInter-Run Intra-Run Total Sample Category N Mean SD % CV SD % CV SD % CVSD % CV SD % CV Low Positive  79* 35.43 0.00 0.00 0.00 0.00 0.22 0.610.49 1.40 0.54 1.52 Medium Positive  79* 34.13 0.00 0.00 0.00 0.00 0.160.48 0.48 1.39 0.50 1.48 Positive Control 80 32.80 0.00 0.00 0.00 0.000.07 0.20 0.59 1.80 0.59 1.81 Negative 80 0.00 Not Applicable NTC** 200.00 Not Applicable *One Replicate was “Invalid” **One Replicate of NTCincluded in each run

Performance of the Assay in Presence of Potentially InterferingSubstances

The performance of this assay was evaluated with potentially interferingsubstances that may be present in stool samples at the concentrationsindicated in the table below. A total of 21 potentially interferencesubstances in replicates of 4 each and baseline (positive) sample inreplicates of 5 were tested initially. All but two interference samplestested as “Positive” in all 4 replicates during initial run. All fivereplicates were “Positive” for two interference substances (Vancomycin &PEPTO BISMOL® bismuth subsalicylate (The Procter & Gamble Company,Cincinnati, Ohio)) upon repeat/confirmatory run. No Interference wasobserved.

Final Sample Preparation C. difficile Substance Active IngredientConcentration Result Mucin Immunoglobulins, 3 mg/mL Detected Lysozyme,Polymers, etc Metronidazole Metronidazole 14 mg/mL Detected VancomycinVancomycin 1.4 mg/mL Detected (8 of 9 replicates) Stearic acid Stearicacid 4 mg/mL Detected Palmitic acid Palmitic acid 2 mg/mL DetectedBarium sulfate Barium sulfate 5 mg/mL Detected Nystatin Nystatin 10,000USP Detected units/mL Whole blood Glucose, Hormones, 5% (v/v) DetectedEnzymes, Ions, Iron, etc. Antacid and Aluminum 0.1 mg/mL DetectedAnti-gas generic Hydroxide (liquid) Magnesium Hydroxide Milk of MagnesiaMagnesium 0.2 mg/mL Detected Hydroxide IMODIUM ® AD Loperamide 0.005mg/mL Detected loperamid (Johnson & Johnson, New Brunswick, NJ) PEPTOBISMOL ® Bismuth 0.175 mg/mL Detected bismuth subsalicylateSubsalicylate (7 of (The Procter & 9 replicates) Gamble Company,Cincinnati, OH) Moist towelettes Benzalkonium 10% (v/v) Detected genericChloride antacid generic Calcium Carbonate 0.1 mg/mL DetectedPreparation H ® Phenylephrine 2% (w/v) Detected ointment (Wyeth, NewYork, NY) Trojan ® spermicide Nonoxynol-9 1.4 mg/mL Detected withnonoxynol-9 (Church & Dwight Co., Princeton, NJ) 1% HydrocortisoneHydrocortisone 2% (w/v) Detected Cream FLEET ® mineral Mineral Oil 2%(w/v) Detected oil (C.B. Fleet Company, Lynchburg, VA) Laxative genericSennosides 0.1 mg/mL Detected ALEVE ® naproxen naproxen sodium 14 mg/mLDetected sodium (Bayer HealthCare, Pittsburgh, PA) K-Y ® Jelly water 2%(w/v) Detected (Johnson & Johnson, New Brunswick, NJ)

Cross Reactivity

Analytical Specificity for various possible cross reactants wasperformed. A total of 47 potential cross reactant organisms were tested.Only the Rotavirus organism tested “Negative” in initial testing of allthree replicates. Confirmatory run of five replicates for “Rotavirus”also tested “Negative”. No cross-reactivity was observed.

Suggested source or Concentration Organism ID tested Dilution ResultAcinetobacter baumannii ATCC 19606 0.5 McFarland 1:1 Did not cross reactAcinetobacter Iwoffii ATCC 15309 0.5 McFarland 1:1 Did not cross reactAdenovirus 40 ATCC VR-931 N/A 1:10⁵ Did not cross react Bacillus cereusATCC 10702 10⁶ CFU/mL 1:860 Did not cross react Bacteroides merdae ATCC43184 0.5 McFarland Neat Did not cross react Bacteroides stercoris ATCC43183 0.5 McFarland Neat Did not cross react Bifidobacteriumadolescentis ATCC 15703 0.5 McFarland Neat Did not cross reactCampylobacter coli ATCC 43479 0.5 McFarland 1:1 Did not cross reactCampylobacter jejuni sub sp ATCC 33292 0.5 McFarland Neat Did not crossreact jejuni Candida albicans ZeptoMetrix 0801504 10⁶ CFU/mL 1:100 Didnot cross react Clostridium tetani ATCC 19406 0.5 McFarland Neat Did notcross react Citrobacter freundii ZeptoMetrix 0801563 10⁶ CFU/mL 1:5200Did not cross react Citrobacter koseri ATCC 27028 0.5 McFarland 1:1 Didnot cross react Clostridium butyricum ATCC 12398 0.5 McFarland Neat Didnot cross react Clostridium difficile (non-toxigenic ATCC 0.5 McFarland1:4500 Did not cross react 700057) Clostridium innocuum ATCC 14501 0.5McFarland Neat Did not cross react Clostridium novyi ATCC 19402 0.5McFarland Neat Did not cross react Clostridium paraputrificum ATCC 257800.5 McFarland Neat Did not cross react Clostridium perfringens ATCC13124 0.5 McFarland Neat Did not cross react Clostridium septicum ATCC12464 0.5 McFarland Neat Did not cross react Clostridium symbiosum ATCC14940 0.5 McFarland Neat Did not cross react Coxsackie virus ATCC VR-3010⁵ TCID₅₀/mL 1:28.1 Did not cross react Cytomegalovirus AD169ZeptoMetrix 0810003CF 10⁵ TCID₅₀/mL 1:20.8 Did not cross react EchovirusATCC VR-36 10⁵ TCID₅₀/mL 1:15.8 Did not cross react Enterobacteraerogenes ZeptoMetrix 0801518 10⁶ CFU/mL 1:10000 Did not cross reactEnterobacter cloacae ATCC 13047 0.5 McFarland 1:1 Did not cross reactEnterococcus faecalis ATCC 51299 0.5 McFarland 1:1 Did not cross reactEscherichia coli ZeptoMetrix 0801517 10⁶ CFU/mL 1:20000 Did not crossreact Fusobacterium varium ATCC 8501 0.5 McFarland Neat Did not crossreact Klebsiella oxytoca ATCC 33496 0.5 McFarland 1:1 Did not crossreact Lactobacillus acidophilus ZeptoMetrix 0801540 10⁶ CFU/mL 1:2120Did not cross react Lactobacillus reuteri ATCC 23272 0.5 McFarland NeatDid not cross react Listeria monocytogenes ZeptoMetrix 0801534 10⁶CFU/mL 1:11800 Did not cross react Norovirus Clinical sample N/A SwabDid not cross react Peptostreptococcus anaerobius ATCC 27337 0.5McFarland Neat Did not cross react Proteus mirabilis ZeptoMetrix 080154410⁶ CFU/mL 1:144 Did not cross react Pseudomonas aeruginosa ZeptoMetrix0801519 10⁶ CFU/mL 1:10500 Did not cross react Rotavirus Clinical sampleN/A Swab Did not cross react Rotavirus (retest) ZeptoMetrix 0810041CF10⁵ TCID₅₀/mL 1:200 Cross reacted* Salmonella enterica subsp. ATCC 140280.5 McFarland 1:1 Did not cross react Enterica (formerly Salmonellacholeraesuis subsp. choleraesuis) Salmonella enterica subsp. ATCC 133140.5 McFarland 1:1 Did not cross react arizonae (Borman) Le Minor et al.deposited as Arizona arizonae Kauffmann and Edwards Serratia marcescensATCC 13880 0.5 McFarland 1:1 Did not cross react Shigella boydii ATCC9207 0.5 McFarland 1:1 Did not cross react Shigella dysenteriae ATCC11835 0.5 McFarland 1:1 Did not cross react Shigella sonnei ATCC 299300.5 McFarland 1:1 Did not cross react Streptococcus agalactiaeZeptoMetrix 0801545 10⁶ CFU/mL 1:22000 Did not cross react Vibriocholerae Genomic DNA 5 ng/μL N/A Did not cross react Negative stoolNA-negative matrix NA-negative N/A Did not cross react matrix*Subsequent testing of the sample at a clinical testing lab confirmedthe sample was positive for C. difficile.

Example 12: Detection of Group a Streptococcus by Direct Nucleic AcidAmplification Assays and Comparison with Methods Using Nucleic AcidExtraction

Nucleic acids from the samples were amplified using the direct nucleicacid amplification assays and the results were compared withamplification results using methods involving nucleic acid extraction.

Swab samples were used in the direct amplification assay. The PCR mastermix was prepared as follows:

PCR Mix

Component Concentration 2.5X Universal MM 1x Scorpion Forward Primer 600nM Reverse Primer 600 nM MgCl₂ 2.5 mM Potassium Chloride 40 mM

2 uL of transport medium from a swab sample was added to 8 uL of PCRmaster mix and PCR was carried out using the following cyclingparameters:

Step Cycles Temp (° C.) Time 1 1 97 6 min 2 40 97 10 sec 60 30 sec

The sequences of the amplification primers and the amplicon are shownbelow. Group A Streptococcus Scorpion primer:

Group A Streptococcus Scorpion primer: 5′d BHQ-1-AGCGGCACTCCAAAAATCAGCAGCTATCAAAGCAGGTGTGCCGC-T(FAM)-Spacer 18-AAGCTTAATATCTTCTGCGCTTC GT 3′(SEQ ID NOS: 28-29) Group A Streptococcus Reverse primer: 5′d TAACCCAGTATTTGCCGATCAA 3′ (SEQ ID NO: 30)Group A Streptococcus amplicon:taacccagtatttgccgatcaaaactttgctcgtaacgaaaaagaagcaaaagatagcgctatcacatttatccaaaaatcagcagctatcaaagcaggtgcacgaagcgcagaagatattaagctt (SEQ ID NO: 31)

The amplification results using the direct nucleic acid amplificationassays were compared with the results obtained using amplificationmethods involving nucleic acid extraction as shown below.

Methods involving nucleic acid extraction % Positive Negative TotalAgreement Direct Posi- 45 3 48 93.8% amplification tive (45/48) methodNega- 3 349 352 99.1% tive (349/352) Total 48 352 400

Thus, 93.8% of the samples identified positive by amplification methodsinvolving nucleic acid extraction also were identified positive by thedirect amplification assay, and 99% of the samples identified negativeby amplification methods involving nucleic acid extraction also wereidentified negative by the direct amplification assay.

Example 13: Detection of Flu a, Flu B and RSV by Direct Nucleic AcidAmplification Assays in the Presence of Interfering Substances

Using the experimental protocol of Example 8, nucleic acid from clinicalspecimens were amplified using the direct nucleic acid amplificationassays in the presence of various interfering substances listed in thetable below:

Interferent Concentration ID Interferent tested Control None N/A 1 humanblood 2% (v/v) 2 AFRIN ® nasal spray 15% (v/v) (Oxymetazoline)(Schering, Kenilworth, NJ) 3 BECONASE AQ ® (Beclomethasone) 5% (v/v)(Glaxo, United Kingdom) 4 Nasal corticosteroid (Fluticasone) 5% (v/v) 5Nasal gel (ZICAM ® nasal gel) 5% (v/v) (Zicam, Scottdale, AZ) 6 Mucin 60μg/mL 7 Systemic antibacterial (Tobramycin) 10 μg/mL 8 RELENZA ®(Zanamivir) 3.3 mg/mL (Glaxo, United Kingdom) 9 TAMIFLU ® oseltamivirphosphate 1.0 μM (Oseltamivir) (Hoffmann-La Roche, Nutley, NJ) 10 Topical antibiotic (Mupirocin) 2.5 mg/mL

The direct amplification assays were conducted in duplicates for eachpotential interfering substance. The Q670 fluorescent label used for aninternal control, PC represents a positive control and NEG represents anegative control.

The table below presents Ct results of detecting the Flu A virus in thepresence of the potential interfering substances.

FAM ™ JOE ™ Interferent fluorophore fluorophore CFR610 Q670 Control 34.80 0 33.5 Control 34.3 0 0 33.3 1 35.2 0 0 33.4 1 35.4 0 0 33.4 2 35.2 00 32.3 2 35.0 0 0 32.4 3 34.5 0 0 33.1 3 34.4 0 0 33.1 4 34.4 0 0 33.2 434.3 0 0 33.2 5 35.6 0 0 33.0 5 34.8 0 0 32.4 6 35.2 0 0 32.8 6 35.2 0 033.2 7 35.7 0 0 33.2 7 34.6 0 0 33.3 8 35.0 0 0 33.5 8 35.1 0 0 33.5 935.2 0 0 33.2 9 34.5 0 0 33.5 10  35.1 0 0 33.3 10  34.8 0 0 33.3 Avg Ct34.9 0.0 0.0 33.1 PC 36.0 33.2 34.4 32.8 NEG 0 0 0 33.4

The table below presents Ct results of detecting the Flu B virus in thepresence of the potential interfering substances.

FAM ™ JOE ™ Name fluorophore fluorophore CFR610 Q670 Control 0 34.7 033.5 Control 0 34.3 0 33.4 1 0 34.9 0 33.4 1 0 34.7 0 33.6 2 0 35.1 032.6 2 0 34.2 0 32.7 3 0 34.6 0 32.7 3 0 34.2 0 32.7 4 0 34.8 0 33.0 4 034.9 0 32.8 5 0 34.5 0 33.4 5 0 34.4 0 33.2 6 0 34.4 0 33.2 6 0 34.4 033.1 7 0 35.2 0 33.1 7 0 34.3 0 33.6 8 0 35.0 0 34.1 8 0 34.4 0 34.0 9 034.9 0 33.3 9 0 34.3 0 33.4 10  0 34.4 0 33.4 10  0 34.3 0 33.3 Avg Ct0.0 34.6 0.0 33.3 PC 35.4 33.4 34.5 32.7 NEG 0 0 0 32.7

The table below presents Ct results of detecting the RSV virus in thepresence of the potential interfering substances.

FAM ™ JOE ™ Name fluorophore fluorophore CFR610 Q670 Control 0 0 34.932.6 Control 0 0 31.4 33.1 1 0 0 34.4 33.6 1 0 0 34.5 33.2 2 0 0 34.932.6 2 0 0 34.9 32.2 3 0 0 34.9 32.4 3 0 0 34.8 33.3 4 0 0 35.1 33.5 4 00 31.0 34.0 5 0 0 34.7 33.3 5 0 0 34.1 33.5 6 0 0 35.1 33.0 6 0 0 34.833.4 7 0 0 35.4 33.9 7 0 0 34.8 34.1 8 0 0 34.9 33.3 8 0 0 35.2 33.6 9 00 34.5 33.8 9 0 0 35.1 33.5 10  38.5* 0 35.1 33.6 10  0 0 34.7 34.0 AvgCt N/A 0.0 34.5 33.3 PC 33.3 33.0 32.7 33.2 NEG 0 0 0 33.0*False-positive FAM ™ fluorophore (Life Technologies) signal wasdetected in 1 of 2 replicates.

The results were verified to be accurate based on a lack of validamplification signal in the FAM™ fluorophores (Life Technologies), JOE™fluorophore (Life Technologies) or CFR610 channels for the negativecontrol and a Ct<40 in the Q670 channel. Also, the PC reactions gave aCt<40 in the FAM™ fluorophores (Life Technologies), JOE™ fluorophore(Life Technologies) and CFR610 channels.

The results of the direct amplification assays for detecting Flu A, FluB and RSV demonstrate that there was no significant change in Ct valueswith any of the interferents for any of the viruses tested, as comparedwith the control samples without any interfering substance. The directamplification assays are therefore not affected by potential interferingsubstances when detecting low positive samples of these viruses.

Example 14: Detection of HSV-1 and HSV-2 by Direct Nucleic AcidAmplification Assays in the Presence of Interfering Substances

Using the experimental protocol of Example 3, nucleic acid on a negativeswab matrix and in synthetic cerebralspinal fluid were amplified usingthe direct nucleic acid amplification assays in the presence of variousinterfering substances listed in the table below:

Interferent Concentration ID Interfering Substance Matrix tested 1 WholeBlood swab and CSF 10% (v/v) 2 Female Urine swab 10% (v/v) 3 Albumin(protein) swab and CSF 10 mg/mL 4 Casein (protein) swab and CSF 10 mg/mL5 K-Y ® Jelly swab 5% (v/v) Johnson & Johnson, New Brunswick, NJ) 6Acyclovir swab and CSF 2.5 mg/mL (Acycloguanosine) 7 BETADINE ® swab andCSF 5% (v/v) microbicide (topical antiseptic) (Purdue Products,Stamford, CT) 8 White Blood Cell CSF 5.5 × 10e8 WBC/mL 9 Hemoglobin* CSF0.625-5.0 mg/mL Control None swab and CSF N/A

The whole blood potential interfering substance (Interferent ID: 1) wastested at 10%, which is clinically more relevant than purifiedhemoglobin. Also, the hemoglobin potential interfering substance(Interferent ID: 9) was tested at higher concentrations of 5.0-1.25mg/mL, but detection of HSV-1 and HSV-2 were inhibited at theseconcentrations.

The direct amplification assays were conducted in triplicates for eachpotential interfering substance. The Q670 fluorescent label was used foran internal control, PC represents a positive control and NEG representsa negative control.

The table below presents Ct results of detecting the HSV-1 virus on thenegative swab matrix in the presence of the potential interferingsubstances.

Interferent HSV-2 HSV-1 IC ID (FAM ™) (CFR610) (Q670) Control 0 33.732.7 Control 0 33.1 32.3 Control 0 32.9 32.2 Control 0 35.8 32.2 1 032.1 31.7 1 0 33.1 31.5 1 0 31.8 31.9  2* 0 30.9 31.1  2* 0 30.8 31.0 2* 0 30.4 30.9 3 0 34.1 31.0 3 0 31.7 31.2 3 0 32.4 31.4 4 0 32.1 31.74 0 32.5 31.1 4 0 32.1 31.5 5 0 31.7 31.2 5 0 31.2 31.6 5 0 31.7 31.6 60 31.5 31.1 6 0 34.2 31.0 6 0 32.7 31.3 7 0 36.1 31.5 7 0 33.4 31.7 7 033.6 32.1 PC (day 1) 31.0 31.2 31.0 NEG (day 1) 0 0 31.1 PC (day 2) 30.531.2 31.0 NEG (day 2) 0 0 31.1

The fluid check for female urine (Interferent ID: 2) failed, asindicated by higher fluorescent values than the control with no sample,although HSV-1 was detected in the female urine samples. The K-Y brandjelly (Interferent ID: 5) produced an earlier Ct value compared tocontrol samples, even though the same HSV-1 levels were used for allpotential interference substance testing. The female urine and K-Y jellypotential interferents were retested and reproducibly produced anearlier Ct for both samples.

The table below presents Ct results of detecting the HSV-2 virus on thenegative swab matrix in the presence of the potential interferingsubstances.

Interferent HSV-2 HSV-1 IC ID (FAM ™) (CFR610) (Q670) Control 33.5 031.6 Control 33.2 0 31.7 Control 33.2 0 32.0 Control 33.7 0 32.1 1 33.30 31.1 1 34.7 0 31.1 1 35.2 0 31.2  2* 33.3 0 31.0  2* 33.1 0 31.0  2*32.9 0 31.2 3 33.1 0 31.6 3 33.2 0 31.0 3 33.1 0 31.3 4 32.9 0 32.0 433.1 0 30.7 4 32.4 0 31.3 5 33.0 0 31.1 5 32.9 0 31.4 5 33.0 0 31.2 632.6 0 31.2 6 33.1 0 31.5 6 32.7 0 31.1 7 33.2 0 31.0 7 33.0 0 31.3 732.8 0 30.9 PC (day 1) 31.0 31.2 31.0 NEG (day 1) 0 0 31.1 PC (day 2)30.5 31.2 31.0 NEG (day 2) 0 0 31.1

The fluid check problem relating to the female urine sample describedabove for HSV-1 also applied to HSV-2.

The table below presents Ct results of detecting the HSV-1 virus in thesynthetic cerebralspinal fluid in the presence of the potentialinterfering substances.

Interferent HSV-2 HSV-1 IC ID (FAM ™) (CFR610) (Q670) Control 0 34.431.9 Control 0 33.5 32.2 Control 0 32.5 32.2 Control 0 34.0 32.3 1 035.0 32.4 1 0 33.3 31.7 1 0 32.9 31.9 3 0 34.2 31.7 3 0 34.4 31.2 3 033.6 31.7 4 0 35.0 32.1 4 0 36.4 31.4 4 0 36.7 31.5 6 0 35.5 32.2 6 033.5 31.4 6 0 34.3 32.5 7 0 33.9 33.1 7 0 36.1 32.6 7 0 33.8 32.9 8 034.4 31.7 8 0 33.4 31.6 8 0 32.1 31.3 9 0 34.1 31.7 9 0 32.6 31.4 9 032.4 32.0 PC (day 1) 31.0 31.2 31.0 NEG (day 1) 0 0 31.1 PC (day 2) 30.531.2 31.0 NEG (day 2) 0 0 31.1

The table below presents Ct results of detecting the HSV-2 virus in thesynthetic cerebralspinal fluid in the presence of the potentialinterfering substances.

Interferent HSV-2 HSV-1 IC ID (FAM ™) (CFR610) (Q670) Control 34.2 032.4 Control 34.2 0 31.9 Control 34.7 0 32.4 Control 34.6 0 32.3 1 34.60 31.5 1 36.0 0 31.5 1 36.3 0 31.2 3 34.3 0 32.3 3 33.5 0 32.1 3 34.5 032.1 4 33.4 0 32.0 4 34.0 0 31.5 4 33.2 0 32.2 6 34.0 0 32.4 6 34.6 031.8 6 33.3 0 31.8 7 34.1 0 31.9 7 35.1 0 31.7 7 34.0 0 32.6 8 33.3 031.6 8 33.6 0 31.4 8 32.6 0 31.9 9 34.0 0 31.8 9 34.0 0 31.2 9 33.1 031.9 PC (day 1) 31.0 31.2 31.0 NEG (day 1) 0 0 31.1 PC (day 2) 30.5 31.231.0 NEG (day 2) 0 0 31.1

The results were verified to be accurate based on a lack of validamplification signal in the FAM™ fluorophores (Life Technologies) orCFR610 channels for the negative control and a Ct<40 in the Q670channel. Also, the PC reactions gave a Ct<40 in the FAM™ fluorophore(Life Technologies) and CFR610 channels.

The results of the direct amplification assays for detecting HSV-1 andHSV-2 demonstrate that there was no significant change in Ct values withany of the interferents for either of the viruses tested. The directamplification assays are therefore not affected by potential interferingsubstances when detecting low positive samples of these viruses.

Example 15: Components of Direct Amplification Assays Providing ImprovedPerformance

The following data represent components of the direct amplificationassays that allow for improved performance. In one embodiment, acombination of all of these components provides a system that allowsreal time PCR to be effective even in the presence of known inhibitors(e.g., blood and heparin).

Effect of KCl

Adding KCl (up to 20-40 mM) to a direct amplification assay improvesfluorescence signals and confers improved sensitivity to the reaction.The data in FIG. 5 demonstrates detection of MRSA in the presence andabsence of KCl. Although some detection is possible without KCl, thepresence of KCl improves the efficacy of the reaction. The data in FIG.5 was generated in the presence of other reaction components, such as acationic surfactant.

Effect of BSA

BSA was added to stool samples containing C. difficile, at variousconcentrations. At higher concentrations, represented by the 3500ng/reaction below, inhibition was removed from some patient samples. Ascan be seen in the table below (presenting Ct values), at a lower 5ng/reaction concentration of BSA, the C. difficile target was notdetected and the internal control was missed in 2 out of 3 samples.Specifically, the internal control was detected with Sample A. However,the lower Ct value, as compared with the sample at higherconcentrations, demonstrates delayed amplification and is characteristicof inhibition (the Ct value is the PCR cycle at which sufficient signalis generated to detect the target in question).

BSA (3500 ng) BSA (5 ng) Internal Internal Sample C. diff Control C.diff Control A 34.30 30.30 0 35.70 B 34.10 31.90 0 0 C 33.90 33.20 0 0

Effect of Surfactants

Adding cationic surfactants improves fluorescence signals and confersimproved sensitivity to a direct amplification assay. As demonstrated inFIG. 6, in which a direct amplification assay was conducted on SimplexaBordetella, some detection is possible without addition of thesurfactant. However, when using the surfactant, efficacy of the reactionis improved, as demonstrated by greater signal height, which confersimproved sensitivity.

Effect of Additional Heating

Some organisms tested require additional heating steps to improve assayperformance. For example, sensitivity improvements were observed for FluB when using additional heating beyond that provided in a standardreaction (e.g., pre-heating), as demonstrated in FIG. 7. The improvementwith Flu B was seen at a temperature of 70° C. Assay performancedetecting other organisms, such as C. difficile and Group AStreptococcus, was seen at a temperature of 95° C. Heating the sample todestroy inhibitors and to lyse organisms must be balanced with thepotential for heat to destroy reagents. In some embodiments, heating thesamples is performed prior to adding the reagents (e.g., buffer).

Tolerance of System

The data below (Bordetella pertussis/parapertussis PCR) shows that thedirect amplification assays can tolerate up to 30% transport media(Copan UTM) without inhibition. All samples tested below with directdetection methodology used a 30% sample and 70% direct amplificationreaction mix. Results from direct detection were compared to resultsusing DNA extraction and purification prior to amplification. The directmethod had 99% sensitivity and specificity compared to the extractionand purification test using patient specimens.

Direct Detection Direct Detection Positive Negative Extracted Method 433 Positive Extracted Method 3 409 Negative

Previous publications required significant dilution prior to addingspecimen to the reaction mixture, therefore limiting sensitivity.

Example 16: Direct Amplification Assays with Additional Specimen TypesWhole Blood

Whole blood was used to perform human genetic testing. Whole blood couldbe used with either a 1:4 dilution or with 0.5 μl in a 10 μl reactionvolume. In either case, the heme (which is a known PCR inhibitor) didnot affect the assay. Complete concordance was achieved when testing forthe presence of Factor V Leiden mutations or Factor II mutations usingthe direct amplification method and when using the reference methodwhich utilized nucleic acid extraction prior to amplification andmutation detection.

Simplexa Direct FVL WT HET HOM Total REF WT 489 0 0 489 HET 0 50 0 50HOM 0 0 4 4 Total 489 50 4 543

Simplexa Direct FII WT HET HOM Total REF WT 519 0 0 519 HET 0 24 0 24HOM 0 0 0 0 Total 519 24 0 543

Whole Blood with Heparin

The data in FIG. 8 shows amplification plots from a single blood samplethat was collected into 3 tubes, each with a different anticoagulant(heparin, EDTA, citrate). As can be seen, the amplification plots showthat all samples give efficient amplification, even when heparin, aknown PCR inhibitor, is used as the anticoagulant.

Buccal Swabs

The data in FIG. 9 represents 4 replicates from each of 2 samples ofhuman genetic DNA for mutations (detecting the Factor V Leiden mutationregion), plus one negative control. Efficient amplification is seen forall replicates. Samples were collected by the swabbing inner cheek forabout 10 seconds to ensure the whole swab surface was used. The swabswere then placed into 500 uL 1×TE Buffer 2 uL, which was loaded directlyinto the PCR sample without extraction.

Comparison to Previously Published Literature

Previously published results indicate that in order for effectivedetection, samples must be diluted. Compared to these publications, forexample, the Pandori et al., BMC Infect. Dis., 6:104 (2006) reference,the foregoing data demonstrates a 10 fold greater amount of sample,providing a limit of detection that is 10 fold lower than the publishedmethod.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the inventions embodied thereinherein disclosed may be resorted to by those skilled in the art, andthat such modifications and variations are considered to be within thescope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed is:
 1. A method for identifying the presence or absence of a target nucleic acid from a microorganism in a biological sample obtained from a human, said method comprising: (a) contacting the biological sample with a reaction mixture containing a DNA polymerase and a buffer to form a sample mixture under conditions suitable for amplification of the target nucleic acid from the biological sample without extracting the target nucleic acid from the biological sample; (b) amplifying the target nucleic acid; and (c) identifying the presence of the target nucleic acid from the microorganism by detecting the amplified target nucleic acid, wherein the biological sample is prepared by mixing a swabbed specimen from the human in a buffer to produce the biological sample, wherein 20-30% of the total volume of the sample mixture following step (a) is the biological sample, wherein the swabbed specimen is from whole blood, plasma, serum, cerebrospinal fluid (CSF), or stool, wherein the nucleic acid in the biological sample is not extracted from the biological sample prior to amplification; wherein the target nucleic acid is not present in a biological sample that does not contain the microorganism; and wherein the reaction mixture contains a cationic surfactant.
 2. The method of claim 1, wherein the biological sample is heated prior to step (a) or step (b).
 3. The method of claim 1, wherein the reaction mixture comprises potassium chloride.
 4. The method of claim 1, wherein the reaction mixture comprises albumin.
 5. The method of claim 1, wherein the microorganism is a virus.
 6. The method of claim 5, wherein the virus is selected from the group consisting of an influenza virus, a respiratory syncytial virus, a varicella zoster virus, a herpes simplex virus, and an enterovirus.
 7. The method of claim 6, wherein the influenza virus is influenza Type A or influenza Type B.
 8. The method of claim 6, wherein the herpes simplex virus is herpes simplex virus 1 (HSV-1) or 2 herpes simplex virus (HSV-2).
 9. The method of claim 1, wherein the microorganism is a bacterium.
 10. The method of claim 8, wherein the bacterium is Clostridium or Streptococcus.
 11. The method of claim 10, wherein the bacterium is C. difficile.
 12. The method of claim 10, wherein the bacterium is Group A Streptococcus. 